Yasuyuki Hashidoko

Suppression of Damping-Off Disease in Host Plants by the Rhizoplane Bacterium Lysobacter sp. Strain SB-K88 Is Linked to Plant Colonization and Antibiosis against Soilborne Peronosporomycetes

ABSTRACT We previously demonstrated that xanthobaccin A from the rhizoplane bacterium Lysobacter sp. strain SB-K88 suppresses damping-off disease caused by Pythium sp. in sugar beet. In this study we focused on modes of Lysobacter sp. strain SB-K88 root colonization and antibiosis of the bacterium against Aphanomyces cochlioides, a pathogen of damping-off disease. Scanning electron microscopic analysis of 2-week-old sugar beet seedlings from seeds previously inoculated with SB-K88 revealed dense colonization on the root surfaces and a characteristic perpendicular pattern of Lysobacter colonization possibly generated via development of polar, brush-like fimbriae. In colonized regions a semitransparent film apparently enveloping the root and microcolonies were observed on the root surface. This Lysobacter strain also efficiently colonized the roots of several plants, including spinach, tomato, Arabidopsis thaliana, and Amaranthus gangeticus. Plants grown from both sugar beet and spinach seeds that were previously treated with Lysobacter sp. strain SB-K88 displayed significant resistance to the damping-off disease triggered by A. cochlioides. Interestingly, zoospores of A. cochlioides became immotile within 1 min after exposure to a SB-K88 cell suspension, a cell-free supernatant of SB-K88, or pure xanthobaccin A (MIC, 0.01 μg/ml). In all cases, lysis followed within 30 min in the presence of the inhibiting factor(s). Our data indicate that Lysobacter sp. strain SB-K88 has a direct inhibitory effect on A. cochlioides, suppressing damping-off disease. Furthermore, this inhibitory effect of Lysobacter sp. strain SB-K88 is likely due to a combination of antibiosis and characteristic biofilm formation at the rhizoplane of the host plant.

The phytochemistry of Rosa rugosa

The phytochemistry of Rosa rugosa is reviewed. The structures of the secondary metabolites identified are listed and aspects of their chemistry are discussed. The metabolites are grouped according to structural classes and include hydrolysable tannins (contained in the leaves and petals), catechin derivatives (roots), flavonoids (leaves), 2-phenoxychromones (leaves), monoterpenes (floral parts, leaves), sesquiterpenes (leaves, especially from glandular trichomes) and triterpenes (leaves and roots). The physiological significance of these metabolites is considered from the perspective of assessing their contribution to the survival and adaptability of the species.

Effects of agricultural land-use change and forest fire on N2O emission from tropical peatlands, Central Kalimantan, Indonesia

Abstract Nitrous oxide (N2O) fluxes from tropical peatland soils were measured at a grassland, three croplands, a natural forest, a burned forest and a regenerated forest in Central Kalimantan, Indonesia. Only croplands received fertilization (665–1278 kg N ha−1 year−1). Mean annual N2O emissions from croplands were 21–131 kg N ha−1 year−1 in 2002–2003 and 52–259 kg N ha−1 year−1 in 2003–2004, and were significantly higher than the emissions from other comparable sites. Cropland N2O emissions were among the highest values reported from cultivated tropical, temperate and boreal organic soils. Mean annual N2O emissions were 7.1 (2002–2003) and 23 (2003–2004) kg N ha−1 year−1 from grassland, and were significantly higher than in natural, regenerated and burned forests (0.62, 0.40 and 0.97 kg N ha−1 year−1 in 2002–2003 and 4.4, 4.0 and 1.5 kg N ha−1 year−1 in 2003–2004, respectively). Annual N2O emissions did not differ significantly between forests in 2002–2003, but were significantly lower in burned forest in 2003–2004. Annual N2O emission was significantly correlated between years. Regression analysis revealed that annual N2O emissions in 2003–2004 were 1.9-fold the corresponding 2002–2003 value (annual precipitation of 2339 and 1994 mm, respectively). N2O fluxes were higher during the rainy season than during the dry season at all sites except the regenerated forest. N2O fluxes in cropland and grassland were significantly lower when the water-filled pore space (WFPS) was less than 60–70%, and increased with an increase in soil NO3–N concentration when WFPS exceeded this threshold. Thus, changes in soil moisture were important in controlling seasonal changes in N2O emission. Our results suggest that changing land use from forestry to agriculture will increase N2O production. The effect of forest fires on N2O emission from these soils was not clear.

Antifungal melampolides from leaf extracts of Smallanthus sonchifolius

Abstract A new antifungal melampolide, 8-angeloyl-1(10),4,11(13)-germacuratrien-12,6-olid-14-oic acid methyl ester, named sonchifolin, as well as three known melampolides, polymatin B, uvedalin and enhydrin, were isolated from leaf extracts of Yacon [ Smallanthus sonchifolius (Poepp. and Endl.) H. Robinson]. Sonchifolin exhibited the highest fungicidal activity against Pyricularia oryzae , a fungus causing rice blast disease and the ED 50 value for the spore germination was 22 ppm. This is the first report of these melampolides as fungicidal compounds.

Deep supercooling xylem parenchyma cells of katsura tree (Cercidiphyllum japonicum) contain flavonol glycosides exhibiting high anti‐ice nucleation activity

Xylem parenchyma cells (XPCs) of boreal hardwood species adapt to sub-freezing temperatures by deep supercooling to maintain a liquid state of intracellular water near -40 degrees C. Our previous study found that crude xylem extracts from such tree species exhibited anti-ice nucleation activity to promote supercooling of water. In the present study, thus, we attempted to identify the causative substances of supercooling. Crude xylem extracts from katsura tree (Cercidiphyllum japonicum), of which XPCs exhibited deep supercooling to -40 degrees C, were prepared by methanol extraction. The crude extracts were purified by liquid-liquid extraction and then by silica gel column chromatography. Although all the fractions obtained after each purification step exhibited some levels of anti-ice nucleation activity, only the most active fraction was retained to proceed to the subsequent level of purification. High-performance liquid chromatography (HPLC) analysis of a fraction with the highest level of activity revealed four peaks with high levels of anti-ice nucleation activity in the range of 2.8-9.0 degrees C. Ultraviolet (UV), mass and nuclear magnetic resonance (NMR) spectra revealed that these four peaks corresponded to quercetin-3-O-beta-glucoside (Q3G), kaempferol-7-O-beta-glucoside (K7G), 8-methoxykaempferol-3-O-beta-glucoside (8MK3G) and kaempferol-3-O-beta-glucoside (K3G). Microscopic observations confirmed the presence of flavonoids in cytoplasms of XPCs. These results suggest that diverse kinds of anti-ice nucleation substances, including flavonol glycosides, may have important roles in deep supercooling of XPCs.

Structure elucidation of xanthobaccin A, a new antibiotic produced from Stenotrophomonas sp. strain SB-K88

Abstract The major antibiotic constituent, xanthobaccin A, was isolated from broth cultures of Stenotrophomonas sp. strain SB-K88 as a major antifungal substance. The structure elucidation by spectroscopic analyses and chemical conversion revealed it to be a new tetramic acid-containing macrocyclic lactam.

Salkowski’s Reagent Test as a Primary Screening Index for Functionalities of Rhizobacteria Isolated from Wild Dipterocarp Saplings Growing Naturally on Medium-Strongly Acidic Tropical Peat Soil

Rhizobacteria isolated from wild dipterocarp saplings in Central Kalimantan, Indonesia, were subjected to Salkowski’s reagent test, which is often used in detecting indolic substances. Among 69 isolates grown in a low-nitrogen medium supplemented with L-tryptophan (TRP), culture fluids of 29 strains were positive to the test, in which 17 bacteria turned red and other 10 pink. All the red type rhizobacteria actively converted TRP into tryptophol (TOL), while some yielded indole-3-acetic acid (IAA) with TOL production. They also showed a capacity to decompose gallotannin into pyrogallol via gallic acid. On the other hand, an active IAA-producing Serratia sp. CK67, and three Fe-solubilizing Burkholderia spp. CK28, CK43, and Citrobacter sp. CK42, were all involved in pink type rhizobacteria, which were more effective, oxidative TRP-degraders than the red type rhizobacteria. Thus, Salkowski’s reagent test should be a useful primary index in the screening of functional rhizobacteria in peatland ecosystem.

Soft gel medium solidified with gellan gum for preliminary screening for root-associating, free-living nitrogen-fixing bacteria inhabiting the rhizoplane of plants.

For preliminary screening for and characterization of free-living nitrogen-fixing bacteria from rhizoplane microflora, we used Winogradskys mineral mixture-based nitrogen-free medium solidified with 0.3% gellan gum. The soft gel medium enabled some reference and wild free-living nitrogen-fixing bacteria to grow in characteristic colonies, including their reaction to oxygen and their motility change. Gellan gum is thus likely to be a better gel matrix than agarose for the investigation of root-associating, free-living nitrogen-fixing bacteria to identify their characteristic behaviors.

Cloning of a DNA Fragment Carrying the 4-Hydroxycinnamate Decarboxylase (pofK) Gene from Klebsiella oxytoca, and Its Constitutive Expression in Escherichia coli JM109 Cells

The 4-hydroxycinnamate decarboxylase gene (pofK gene) was cloned from Klebsiella oxytoca, an epiphytic bacterium able to decarboxylate hydroxycinnamic acids to styrene derivatives, in Escherichia coli JM109. Colonies of the enzyme activity-positive transformants were screened by a selection assay combined with an antifungal test using Cladosporium herbarum IHU9262 as the bio-indicator. Two positive transformants constitutively producing 4-hydroxycinnamate decarboxylase were obtained. One of the transformants had a recombinant plasmid designated as pTCD100 in which a 9.6 kb HindIII segment carrying pofK gene was contained. As the expression of the pofK gene in K. oxytoca was substrate-inducible, it was most likely that the pofK gene expression in E. coli cells was free from any regulation by a pofK gene repressor postulated in K. oxytoca cells. The decarboxylase synthesized in E. coli cells showed almost the same specific activity as that of K. oxytoca induced by the substrate.

Antimicrobial sesquiterpene from damaged Rosa rugosa leaves

Abstract An antimicrobial sesquiterpene has been isolated from diffusates of damaged leaves of Rosa rugosa . Its structure, including relative stereochemistry, was deduced by chemical and spectroscopic (UV, IR, NMR and mass spectrometry) methods to be a novel carotane with an α,β-unsaturated aldehyde group, an endo -peroxide bridge and an allyl alcohol partial structure. The corresponding carboxylic acid was found in intact leaves of R. rugosa , but the compound is non-fungitoxic.